Chapter

Natural Products in Drug

Discovery
Akshada Amit Koparde, Rajendra Chandrashekar Doijad
and Chandrakant Shripal Magdum

Abstract

Drug discovery using natural products is a challenging task for designing new
leads. It describe the bioactive compounds derived from natural resources, its
phytochemical analysis, characterization and pharmacological investigation. It
focuses on the success of these resources in the process of finding and discover-
ing new and effective drug compounds that can be useful for human resources.
From many years, natural products have been acting as a source of therapeutic
agents and have shown beneficial uses. Only natural product drug discovery plays
an important role to develop the scientific evidence of these natural resources.
Research in drug discovery needs to develop robust and viable lead molecules,
which step forward from a screening hit to a drug candidate through structural
elucidation and structure identification through GC–MS, NMR, IR, HPLC, and
HPTLC. The development of new technologies has revolutionized the screening
of natural products in discovering new drugs. Utilizing these technologies gives us
an opportunity to perform research in screening new molecules using a software
and database to establish natural products as a major source for drug discovery. It
finally leads to lead structure discovery. Powerful new technologies are revolution-
izing natural herbal drug discovery.

Keywords: natural products, herbal, drug discovery, phytochemicals, bioactive

1. Introduction

Natural products and traditional medicines are of great importance. Natural

products and their derivatives have been recognized for many years as a source of
therapeutic agents and structural diversity. Natural products have a wide range of
diversity of multidimensional chemical structures; in the meantime, the utility
of natural products as biological function modifiers has also won considerable
attention [1].
Drug discovery is leading to be a challenging scientific task to find robust and
viable lead candidates, which is nothing but the process flow from a screening of
natural product to a new isolate that requires expertise and experience. However,
in addition to their chemical structure diversity and their biodiversity, the develop-
ment of new technologies has revolutionized the screening of natural products in
discovering new drugs [2]. Applying these technologies offers a unique opportunity
to reestablish natural products as a major source for drug discovery. The present

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article attempts to describe the process of isolation, characterization, and utiliza-

tion of bioactive compounds derived from natural products as drug candidates
called as lead, which focus on the success of pharmacological activity in the process
of finding new and effective drug compounds; this process is commonly referred to
as “natural product in drug discovery.”
Natural products played a vital role on this earth, so man’s existence has been
made possible. The outstanding phenomenon of nature always stands as golden
mark for achieving the herbal drug discovery [3].
From earlier decades medicinal plants existed on earth. Thus, medicinal herbs
are of global and paramount importance. The world is decorated with medicinal
herbs, which is a rich wealth of endurance. Every plant is identified by its own dif-
ferent therapeutic properties due to active bioactive molecule. In the modern system
of medicine, natural drug substances are reported to be vital and have appreciable
roles. Their therapeutic role was justified by the presence of their bioactive mol-
ecules. Due to disease-inhibiting capabilities, they are extremely useful as natural
drugs, provide basic bioactive compounds that are less toxic and more effective,
and bring biological and chemical means of modification and extraction of natural
products into potent drug.
The raw materials for Ayurvedic medicines were mostly obtained from plant
sources in the form of crude drugs such as dried herbal powders or their extracts
or mixture of products. Apart from these systems, there has been a rich heritage
of ethnobotanical usage of herbs by various colorful tribal communities in the
country [4].
It has been estimated that nearly 75,000 species of higher plants exist on the
earth, and only 10% have been used in traditional medicine. Only 1 to 5% have been
studied scientifically and are known to have therapeutic value [5].
Around the globe, herbal medicine is based on traditional medicine. As per the
oral survey made in many regions of the world, it has been said that traditional
medicines have their own importance and basic philosophy. So exploration of the
chemical constituents of the plants and their pharmacological screening may pro-
vide us the basis for developing a lead molecule through herbal drug discovery. The
very important life-saving drugs have been provided by herbs in modern medicine.
But among the estimated 4-lakh plant species, only 6% have been studied for their
activity and very less not more than of 20% have been investigated phytochemically
[6]. Thus, there is a need of investigating the various bioactive fractions and the
phytoanalysis and phytopharmacological evaluation of herbal drugs for achieving
the dreams of herbal drug discovery.
Working role of every green herbal drugs from plant source and synthesis of
bioactive products in their own way as God’s gift and preserve them within which
are extractable and used raw material as and when required through various
scientific process for various scientific investigations and study of herbal drug
discovery. Many pharmaceutical compounds contain secondary metabolites of
plants that are of vital importance in drug designing. However, in order to have
a good supply of the source material, some factors like environmental changes,
diverse geographical distribution, labor cost, and selection of the superior
plant should be taken care of by green plant developers so that good plants
will be beneficial to pharmaceutical industry to develop good-quality herbal
drugs [7].
Natural products have played, and will continue to play, a key role in drug discov-
ery and are therefore traditionally claimed as the cornerstones of drug discovery and
development. Many drugs that are available in market today were discovered from
natural sources [8]. An important example is the analgesic activity of aspirin [22],

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which is so far the world’s best known and most universally used medicinal agent.
Its origin is from the plant genera Salix spp. and Populus spp. and it is related to
salicin. A good example is serendipitous discovery of the antibiotic penicillin [22] in
the laboratory from the fungus Penicillium notatum.

Aspirin [22] Penicillin [22]

Many other examples show the value and importance of natural products from
plants and microorganisms in modern days. Paclitaxel (Taxol [22]), which was first
isolated from the bark of the Pacific yew tree Taxus brevifolia (Taxaceae), is the
most recent example of an important natural product that has made an impact in
medicine. Activity against a variety of retroviruses, including HIV, two compounds
isolated from Hypericum perforatum (Guttiferae) are hypericin and pseudohy-
pericin. They are of paramount importance due to inhibition of release of reverse
transcriptase by stabilizing the structure of the HIV capsid and thus preventing the
uncoating process [9, 10].

Taxol [22]

In four different ways, medicinal plants having good therapeutic properties are
valuable for modern system of herbal and natural drug discovery.

1. They are used as direct sources of therapeutic and bioactive agents.

2. Bioactive fractions serve as raw material base for the elaboration and devel-
opment of herbal-based more complex semisynthetic chemical compounds.

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Figure 1.
Various strategies for the discovery of drugs from natural resources.

3. The isolated structures derived from herbal plant species can be used as lead
for new drug discovery in developing herbal compounds.

4. Lastly, plants can be used as bioactive markers for the spectroscopic and
chromatographic analysis along with the discovery of new compounds.

Various strategies for the discovery of drugs from natural resources can be seen
in Figure 1.

2. I mportant steps for successful completion of natural

drug discovery

Phytochemistry or phytoanalysis of natural product in chemistry research is the

backbone and pillar of herbal pharmaceutical as well as food industry. To achieve
success in natural drug discovery and use of herbals in modern medicine, the steps
to be followed are listed below [11]:

1. Extraction, isolation with chromatographic separation, purification, and

characterization of new phytoconstituents having good bioactivity

2. Use of newly isolated phytoconstituents as “lead” compound for designing of

new analogues with either improved therapeutic activity or reduced toxicity

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3. Conversation of lead phytoconstituents into medicinally important drugs

by herbal drug discovery and herbal drugs used by common people showing
socioeconomic benefit

3. Practical outlook of herbal drug discovery

The following facets represent outlook of the stages involved in the development
of bioactive molecule as pure drug from a plant source [12].

1. Collection and identification of the plant, authentication, and deposition of

sample in herbarium like the botanical survey of India

2. Literature survey and analysis on the plant species along with the activity
present in the selected plants for studies

3. Extraction of nonpolar to polar solvent and preparation of extracts for phyto-

chemical analysis and their biological testing [13]

4. Evaluation of plant extracts by judging of different biological test methods

5. Chromatographic analysis by activity-guided fractionation of the extract,

monitoring each chromatographic fraction, its isolation calculating Rf
values,area as per the computer based software’s and comparison with avail-
able bioactive markers which leads to the investigation

6. Structure elucidation using spectroscopic techniques of bioactive isolates using

chemical methods

7. Testing of each bioactive compound in all in vitro and in vivo phytopharma-

cological test methods, in order to determine potency and selectivity of the
herbal extract or isolates for the discovery of herbal drugs

8. Performing molecular modeling studies and preparing derivatives of the active

compound of interest

9. When total synthesis is not practical, carrying out large-scale reisolation of

interesting active compounds for toxicological and pharmacological studies

10. Clinical trials (phase I–III).

First of all, in order to study medicinal plants, selection of plant and which type
of pharmacological activity is to be studied should be clear to the researcher. Five
principles of selection of plants are very important to know which are the random,
the taxonomic, the phytochemical, the ethno-medical and the information-man-
aged approach (Figure 2)[14].

• In the random selection, collection of all available plants in the area, which is
to be studied, is collected based only on visualization and observation without
having knowledge and experience about the selected plants.

• In the taxonomic approach, prior knowledge about the plants of interest with
their specific genus or family and their different locations should be known.

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Figure 2.
General procedure for obtaining active principles from plants.

• The phytochemical (chemotaxonomic) approach is based on the knowledge

of bioactive chemical type for treating particular disease of interest should be
known and are collected. Taxonomic and the phytochemical approaches are
interrelated.

• In the ethnomedical approach, selection is totally based on the information of

the medicinal use of that particular plant in various areas.

• Lastly, information managed approach is basically collection of plants based on

survey and use of plants from their local area that gives prior idea about their
usage and activity and then their evaluation scientifically.

4. Position of herbal drugs

In the current era, new and newer diseases are causing threat to common people
around the world. Thus, disease percentage differs in every part of the world, but dis-
eases are not new; due to global warming, they are detected newly. Prevention is better
than cure, so WHO had taken the vouch of providing “Health for all” by 2000 AD.
Multidisciplinary research on plants has led to many new drugs, as well as
prototype active molecules and biological tools; for examples, see [14].

5. Natural drugs available in market: anti-inflammatory

5.1 Himalaya Boswellia

Himalaya herbals are developed herbal product from Boswellia, which are a
pure herb extract. The bioactive molecule constituent in the gum resin of Shallaki
or Boswellia serrata was boswellic acid. Pyrazoline as a lead molecule is present in
boswellic acid. It acts through the mechanism of supporting the body’s natural

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immune response and preventing inflammation and providing healthy joints and
muscle. Boswellia is a natural and safe herb for joint health, as it gently cares for
it. Boswellia is a good promoter of healthy cholesterol and triglyceride levels and
provides broad health and immune-modulating benefits. Boswellia has been used
extensively in Ayurveda for arthritis and to provide an overall sense of well-being.

5.2 Ginger

From long years ago, herbal medicine has paid hats off to ginger due to its ability
to boost the immune system. It is believed that ginger is used in day-to-day life
because it plays an important role in warming the body. It can help to clean our body
from accumulated toxins by its break down in your body. It’s also known to cleanse
the lymphatic system, our body’s sewage system. Ginger prevents the accumula-
tion of toxins and a person’s body is highly safe guarded from viral, fungal, and
bacterial infections. Medicinal plant ginger also shows many health benefits. It is
specially used as natural remedy for nausea and pain alleviation and for its anti-­
inflammatory properties and inhibiting diabetes.

5.3 Licorice root

Licorice is becoming evident and lighten up in various researches for treatment

and prevention of diseases like hepatitis C, HIV, and influenza. From a study, it con-
firms the antiviral activity of licorice root due to its triterpenoid content. It notes
that licorice’s antioxidant, free radical-scavenging and immuno-stimulating effects.
Licorice root benefits also include pain relief.

5.4 Olive leaf

The olive leaf has antiviral properties, giving it the ability to treat the common
cold and dangerous viruses.

5.5 Oregano

Oregano oil benefits are lightening up to be more superior to some antibiotics,

with no harmful side effects on health, and can be used in day-to-day life. Carvacrol
and thymol are the bioactive molecules isolated and studied and reported to have
powerful properties and uses. They act upon viral infections, as well as allergies,
tumors, parasites and disease-causing inflammation.

6. Future avenues in herbal drug discovery

In the current era, in many developed countries, priorities has been given to scien-
tific research on medicinal plants is growing need of an hour in various research insti-
tutes, universities and pharmaceutical laboratories as well as in the clinics thereof.
This research is put forward in mainly two directions: first, bioactive molecule of
plants that have long been known and used for their healing properties based on the
prior knowledge of the survey and literature. The second phase of basic research
has led to the discovery of new medicinal plants with new bioactive molecules, new
bioactivity, and new drugs from the more remote regions of the world [15].
Drugs of Ayurveda, Unani, and Siddha need scientific investigation of each and
every traditional medicine, which should be put forward for testing and valida-
tion. Many government and private companies like CSIR, New Delhi, are already

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involved in this filed and have validated about thousands of formulations for
different activities. This is a welcome trend and it plays a vital role to correlate the
traditional practice with modern knowledge for the betterment of health. WHO
has emphasized the need to ensure the quality control of herbs and herbal formula-
tions by using modern techniques. Almost many countries have their own herbal
pharmacopeias and make time to time amendments for new monographs and
procedures to maintain their quality of herbal products that are benefited by com-
mon man. Example Ayurvedic pharmacopeia of India includes many basic quality
parameters, isolation techniques, separation, and spectroscopic identification for
more than hundred common herbal drugs.

6.1 Analytical methodologies

It plays an eminent role in herbal natural drug discovery, and without analytical
methodologies, it is hardly impossible. Spectroscopic characterization is the back-
bone and pillar of herbal drug discovery. The knowledge of this plays an important
role in developing the new lead, which can be used for designing new molecules
with short modification. The important steps are the extraction, isolation, and
characterization of active ingredients from herbal plants [16]. Different techniques
of extraction are well known as extraction is the most important step toward the
analysis of bioactive constituents. Microwave-assisted extraction and conventional
extraction should be studied specifically, which give the ideas about the yield
obtained. Further, it highlights the isolation of active molecules by chromatographic
techniques like TLC, column chromatography. The most important step toward
analysis of bioactive compounds present in the plant extracts is characterization,
which includes phytochemical screening assays that give ideas about the presence
of secondary metabolites used to cure the health problems. Highly sophisticated
techniques for structure identification of lead molecule bioactive fraction are
high-performance liquid chromatography (HPLC), high-performance thin-layer
chromatography (HPTLC), Fourier-transform infrared spectroscopy (FTIR),
nuclear magnetic resonance (NMR), and gas chromatography–mass spectrometry
(GC–MS). These techniques are the heart and key challenges in research of natural
drug discovery giving rise to natural products in drug discovery.

6.2 Isolation, identification, and characterization of phytochemicals

The combination of various types of bioactive compound or phytochemicals is

usually present in different plant extracts. The different bioactive compounds have
different polarities. Separation, identification, and characterization of bioactive
compounds are a big challenging job in the herbal drug development process.

6.3 Phytochemical screening assay

Phytochemical screening assay is a simple, quick, and inexpensive procedure

that tells about various types of phytochemicals in a mixture and an important tool
in bioactive compound analyses. Phytochemical examinations are carried out for all
extracts as per the standard methods [15].

6.3.1 Tests for carbohydrates

Preparation of test solution: the test extract was prepared by dissolving with
water. Add 1 volume of 2 N HCl so that it gets hydrolyzed and is further subjected
to the following chemical tests:

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• Molisch’s test (general test): take 2 ml of extract, add two drops of α-naphthol
solution in the alcohol and shake, and then add five drops of concentrated H2SO4
to the sides of the test tube to observe violet ring at the junction of two liquids.

• Fehling’s test: in a test tube, add 1 ml Fehling’s A and 1 ml Fehling’s B solutions

and mix and boil for 1 min. Add equal volume (2 ml) of test solution. Heat in
boiling water bath for 5 min. Observe for yellow and then brick red precipitate.

• Benedict’s test: add 1 ml of Benedict’s reagent and 1 ml of test solution in a

test tube and then mix well. Heat in boiling water bath for 5 min. The solution
may appear green, yellow, or red depending on the amount of reducing sugar
present in test solution.

• Barfoed’s test: add 1 ml of Barfoed’s reagent and 1 ml of test solution in the test
tube. Heat for 1–2 min, in boiling water bath, and cool. Observe for red precipitate.

6.3.2 Tests for proteins

• Biuret test (general test): for a 2 ml test solution, add two drops of 4% NaOH
and two drops of 1% CuSO4 solution, and observe for violet or pink color.

• Millon’s test (for proteins): add 2 ml of TS and mix with 4 ml of Millon’s

reagent; observe for white precipitate. Precipitate if warm, turns brick red or
precipitate dissolves giving red color.

• Xanthoproteic test (for protein containing tyrosine or tryptophan): mix 2 ml

TS with 0.5 ml concentrated H2SO4, and observe for white precipitate.

6.3.3 Tests for amino acids

• Ninhydrin test (general test): add 2 ml TS and two drops of 5% ninhydrin solu-
tion, and heat in boiling water bath for 5 min. Observe for purple or bluish color.

• Test for tyrosine: add 2 ml TS and two drops of Millon’s reagent. Heat the solu-
tion and observe for dark red color.

• Test for tryptophan: add 2 ml of TS and 2 drops of glyoxylic acid and concen-
trated H2SO4 and observe for reddish violet ring at the junction of the two layers.

• Test for cysteine: add 2 ml of TS and few drops of 40% sodium hydroxide and
10% lead acetate solution. Boil. Black ppt. of lead sulfate is formed.

6.3.4 Tests for steroid and triterpenoid

• Salkowski reaction: add 3 ml of extract, 3 ml of chloroform, and 3 ml of

concentrated H2SO4 in a test tube; shake well; and observe whether chloroform
layer appeared red and acid layer showed greenish yellow fluorescence.

• Liebermann-Burchard reaction: add 2 ml extract with 2 ml of chloroform and

add 1–2 ml acetic anhydride and 2 drops of concentration H2SO4 from the side
of test tube. Observe for first red, then blue, and finally green color.

• Liebermann’s reaction: add 3 ml of extract with 3 ml acetic anhydride. Heat

and cool. Add few drops of concentrated H2SO4 and observe for blue color.

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6.3.5 Tests for glycosides

Preparation of test solution: the test solution was prepared by dissolving extract
in the alcohol or hydro-alcoholic solution.

a. Tests for cardiac glycosides:

• Baljet’s test: add a test solution with 1 ml of sodium picrate and observe for
yellow to orange color.

• Legal’s test (for cardenolides): to 1 ml of test solution, add 1 ml pyridine and

1 ml sodium nitroprusside. Observe for pink to red color.

• Test for deoxysugars (Keller-Kiliani test): to 2 ml extract, add 0.5 ml glacial

acetic acid, one drop of 5% FeCl3, and concentrated H2SO4. Observe for
reddish brown color at the junction of the two liquid and upper layers bluish
green.

• Liebermann’s test (for bufadienolides): add 2 ml extract to 2 ml acetic

anhydride. Heat and cool. Add few drops of concentrated H2SO4 and observe
for blue color.

b. Tests for saponin glycosides:

• Foam test: the extract was mixed with water and shaken vigorously.
Persistent foam was observed.

• Hemolytic test: add test solution to one drop of blood placed on the glass
slide. Hemolytic zone appears.

c. Tests for anthraquinone glycosides:

• Borntrager’s test: to 3 ml extract, add dil. H2SO4. Boil and filter. To cold
filtrate, add equal volume benzene or chloroform. Shake well. Separate the
organic solvent. Add ammonia. Ammoniacal layer turns pink or red.

• Modified Borntrager’s test: to 3 ml extract, add 3 ml 5% FeCl3 and 3 ml dil.

HCl. Heat for 5 min in boiling water bath. Cool and add benzene, shake well,
and separate organic layer. Add equal volume dil. ammonia in organic layer.
Ammoniacal layer shows pinkish red color.

6.3.6 Tests for flavonoids

Flavonoids are present in hydrolyzed plant extracts. Its presence is maximum

in parts of the leaves and they are highly soluble in methanol. The flavonoids are
all derived structurally from the important substance called flavone. The flavo-
noids occur in the free form as well as bound to sugars as glycosides. Flavonoids
are found maximum in herbal plants and have good phytopharmacological
activities.
Preparation of test solution:

i. To 1 ml of extract, equal volume of 2 M HCl was added and heated in a test

tube for 30 to 40 min. at 100°C.

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ii. The cooled extract was filtered, and extracted with ethyl acetate.

iii. The ethyl acetate extract was concentrated to dryness and used to test for
flavonoids.

• Shinoda test: to 2 ml of extract, add 5 ml of 95% ethanol, 5 drops of concen-

trated HCl, and 0.5 g magnesium turnings. Pink color was observed. To small
quantity of residue, acetate solution was added and observed for yellow col-
ored precipitate. Addition of sodium hydroxide to the residue showed yellow
coloration, which was decolorized after addition of dilute hydrochloric acid.

• Ferric chloride test: to 2 ml of test solution, add few drops of ferric chlo-
ride solution, which shows intense green color.

• Alkaline reagent test: 2 ml of test solution was treated with 2 ml of sodium

hydroxide solution, which showed intense yellow color that became color-
less on addition of few drops of dilute hydrochloric acid.

• Lead acetate solution test: 2 ml of test solution with few drops of lead acetate
solution (10%) gives yellow precipitates.

6.3.7 Tests for alkaloids

• Mayer’s test: test solution treated with Mayer’s reagent (potassium mercuric
iodide); cream colored precipitate was not obtained.

• Wagner’s reagent: the test solution treated with Wagner’s reagent (iodine in
potassium iodide); brown precipitate was not obtained.

• Hager’s test: the test solution treated with Hager’s reagent (saturated picric acid
solution); gives yellow precipitate.

• Dragendorff ’s test: the test solution treated with Dragendorff ’s reagent (potas-
sium bismuth iodide); reddish brown precipitate was not obtained.

6.3.8 Tests for tannins and phenolic compounds

To 2–3 ml of extract, add few drops of the following reagents:

5% FeCl3 solution shows deep blue-black coloration.
Addition of lead acetate solution shows white precipitate.
Addition of gelatin solution shows white precipitate.
Addition of bromine water shows decoloration of bromine water.
Addition of acetic acid solution shows red colored solution.
Addition of dilute iodine solution shows transient red color.
Addition of dilute HNO3 shows reddish to yellow color.
Addition of dilute KMnO4 shows disappearance of Pink color.

7. Chromatography techniques

Chromatography is a technique where the molecules are separated based on their

shape, size, and charge. In any extract, there are hundreds of unknown components
and many of them are in very low amount. During chromatography, analyte in

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solvent and move through solid phase that acts as a sieving material. As molecule
proceeds further through molecular sieve, it gets separated. Moreover, there usually
exists variability within the same herbal materials. Hence, it is very important to
obtain reliable chromatographic fingerprints that represent pharmacologically
active and chemically characteristic components of the herbal medicine. Thin layer
chromatography is a chromatographic technique that readily provides qualitative
information and through which it becomes possible to obtain quantitative data [17].

7.1 Thin layer chromatography (TLC)

Stahl gave the first practical application of thin layer chromatography. TLC is a most
versatile technique and it shows its separation with good speed. Advantage of TLC is
its sensitivity. TLC works on the principle of an adsorption chromatography in which
samples were separated. Separation is based on the interaction between a thin layer of
adsorbent attached on the plate and solvent system. The technique is mostly used for the
separation of low molecular weight compounds. Many different adsorbents are used in
TLC like silica gel, aluminum, cellulose powder, starch, etc. and can be used to separate
various compounds like amino acids, alkaloids, phenols, steroids, vitamins, etc.
It is being implemented extensively due to the following reasons:

1. It carries out good speedy separation and rapid analysis of herbal extracts.

2. It shows with minimum sample clean-up requirement.

3. It has the ability for calculating qualitative and semiquantitative information

of the separated compounds with Rf values.

4. It enables the quantification of chemical constituents (Table 1).

7.2 High performance thin layer chromatography (HPTLC)

HPTLC is a more powerful separation tool for quantitative analysis and it uses
the technique in a more optimized way. High performance thin layer chromatogra-
phy (HPTLC) is based on the principle of planar chromatography where separation
of sample components is achieved on high performance layers with detection and
data evaluation. These high performance layers on TLC plates are precoated with
an adsorbent of 6 micron particle size and a 160 microns layer thickness. The lesser
the thickness of layer and particle size results in increased plate efficiency as well
as nature of separation. HPTLC has an ability to show its performance on graphical
representation in the form of chromatogram. Separation can be easily visualized by
pictorial representation, which is possible only in case of HPTLC. The procedure
used is as follows: silica gel 60 F254 precoated plates (20 × 10 cm) are used with any
developed solvent system. Different extracts are to be spotted on precoated HPTLC
plates. Spots of different concentration (l μL) was applied on HPTLC plates to study
the exact separation of spots. Saturation time will be 20 min and room temperature
25°C ± 2°C. TLC plates were developed up to 8 cm. After air drying, a plate was
heated at 110°C for 2–3 min. In TLC fingerprinting analysis, the information can
be stored and recorded using specific highly sophisticated instruments like high
performance TLC scanner. It gives information about the chromatogram, retarda-
tion factor (Rf) values, the color of the separated bands, their absorption spectra,
and λ max. After derivatization and using different visualization reagents, snaps
of TLC plates can be obtained and saved for further process. Thus, this represents
TLC fingerprint profile of the provided sample. The information so generated has

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Plant constituents Stationary Mobile phase Detection

phase

Carbohydrates Silica gel Ethyl acetate:toluene(1:1) 10% ethanolic

sulfuric acid

Alkaloids/phenanthrenes Silica gel Toluene:ethyl Dragendorff reagent

acetate:diethylamine(7:2:1)

Flavonoids Silica gel Ethyl acetate:formic acid:glacial UV 254 nm or

acetic acid:water(10:1.1:1.1:2.6) 366 nm

Tannins Silica gel Ethyl acetate:formic acid:glacial Vanillin sulfuric acid

acetic acid:water(7.5:0.3:0.2:2) reagent

Saponin glycoside Silica gel Chloroform:glacial acetic acid:met Vanillin sulfuric acid
hanol:water(6.4:3.2:1.2:0.8) reagent

Specific Mobile phases

Betasitosterol Silica gel Benzene:ethylacetate(9:1) Vanillin sulfuric acid

reagent

Rutin Silica gel Ethyl acetate:formic acid:glacial UV 254 nm or

acetic acid:water(10:1.1:1.1:2.6) 366 nm

Curcumin Silica gel Chloroform:methanol(9.8:0.2) Visible light

Gingerol Silica gel Toluene:ethylacetate(9.3:0.7) Vanillin sulfuric acid

reagent

Stigmasterol Silica gel Petroleum ether:ethyl acetate(7:3) Vanillin sulfuric acid

reagent

Table 1.
TLC mobile phase for important classes of phytoconstituents [15].

a potential application in the identification of an authentic drug when compared

with bioactive marker and it helps in maintaining the quality and consistency of the
isolates or herbal drugs in natural drug discovery [18].

7.3 Column chromatography (CC)

Column chromatography works on the principle of ion exchange, molecular

sieves, and adsorption phenomenon. CC is a most useful technique for separation of
active constituents with larger concentration. Sometimes fractions require another
step for concentration. Displacement chromatography is a newer method that
contains elution of bioactive compounds that have great affinity for the adsorbent.
Fractions of elute materials can be more concentrated than the original solution
placed in column. The column was prepared using silica for column chromatography.
The fraction was dissolved in smallest possible volume of solvent and it was mixed
with 2 gms of silica for column chromatography. Wet column or dry column packing
can be done. Packing of column plays an important role in good quality separation.
The mixture was dried to obtain free flowing powder and it was added to column.
Then, the column was eluted with solvent of various proportions. Every eluent was
collected in properly cleaned test tube separately for further studies to be carried out.

7.4 High performance liquid chromatography (HPLC)

High performance liquid chromatography (HPLC) plays a mandatory role in

isolation of natural products. It is a versatile and widely used technique for the isola-
tion and identification. In the modern era, HPLC technique is becoming popular for
studying separation, identification, and fingerprinting study for the quality control

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of herbal plants. Currently, this technique works as the main choice for research
scientists. The multicomponent samples on both an analytical and preparative scale
can be separated and studied more easily by HPLC. Thus resolving power of HPLC
is ideally used for the rapid processing of herbal extracts. HPLC instruments are
designed in modular ways and they contain delivery pump for solvents and manual
injection valve along with an auto-sampler. As sample is introduced in autosample,
it carries toward the important part or heart of HPLC that is an analytical column,
a guard column. Further, a detector, recorder, and printer are used to show a
graphical representation on the software based or installed computer device. In
every chemical separation, the working and result production differ due to the
fact that certain compounds have different migration rates, which can be fulfilled
using HPLC by utilizing a particular column and mobile phase as per the require-
ment for separations. Trial and error concept is applied for developing new mobile
phase along with prior knowledge of separation, its structure, and required solvent
polarity or nonpolarity. Thus, the extent or degree of separation is based upon
the choice of stationary phase or mobile phase. Generally, the identification and
separation of phytochemicals can be achieved by using isocratic system that is using
single mobile phase. Gradient elution sometimes can be used in which the propor-
tion is altered from organic solvent to water. It also depends on time, and it may be
desirable if more than one sample component is studied. Or it also differs from each
other significantly in retention of components with column as per the conditions
achieved. Identification of compounds by HPLC is a crucial part of HPLC assay.
Identification of any bioactive compound by HPLC selection of detector is again
the next important step. Once the detector is selected and the setting is done, the
assay may be developed by trial and error of solvent system. Once the sharpness
of the peak of known sample is obtained, the solvent system can be selected. The
important parameters of this assay is that a clean sharp peak of the known sample
is observed from the chromatograph. The reasonable retention time of identifying
peak should be there. The extraneous peaks at the detection levels should be well
separated from the main sharp peak. At maximum time, UV detectors are popularly
used in HPLC detection. UV detectors are used among all the detectors because
they have high sensitivity and UV absorbance of majority of naturally occurring
compounds is possible at low wavelengths of 200–210 nm. If bioactive compound
needed to be isolated is only present in small amounts within the sample, then the
high sensitivity of UV detection is a bonus in herbal natural drug discovery. Liquid
chromatography coupled with mass spectrometry (LC/MS) is also a powerful tech-
nique for the analysis of complex botanical extracts. It offers accurate determination
of molecular weight of proteins, peptides. Isotopes pattern can also be detected by
this technique. A recent advance includes electrospray, thermospray, and ionspray
ionization techniques, which offer unique advantages of high detection sensitivity
and specificity [19].
HPLC when combined with mass spectrometry (MSn) gives lot of informa-
tion for structural elucidation of the compounds because its ability of recognition
increases and separation with structure identification becomes very easy. Therefore,
when a biomarker which is of a pure standard is unavailable, fast and accurate
identification of bioactive chemical compounds in medicinal herbs is possible
due to the combination of HPLC and MS. In order to count the overall success of
natural product in isolation and separation, the most important is processing of
raw material further to provide a sample suitable for HPLC analysis. The significant
bearing is on the choice of solvent for sample active compounds identification.
The source material that is dried powdered herbal plant material should be studied
very efficiently in earlier stages and steps: first, its dried form and second to learn
about its chemical structural part that is the powder’s ability to release the bioactive

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compound of interest into the solution. In such cases, mobile phase development
initially using TLC and having idea about the solvent system before applying to
HPLC saves your time. Thus, in normal case of extraction, dried plant material is
treated with an organic solvent methanol, chloroform. After extraction, extracts are
dried over rotary evaporator and powdered extracts are concentrated and injected
into HPLC for separation and analysis. HPLC is useful for compounds that cannot
be vaporized or that decompose under high temperature, and it provides a good
complement to gas chromatography for detection of compounds.

8. Methods of detection

8.1 Fourier-transform infrared spectroscopy (FTIR)

FTIR has proven to be a valuable tool for the characterization and identification
of compounds or functional groups (chemical bonds) present in an unknown mix-
ture of plant extracts. It helps for identification and structure determination of the
molecule. In addition, FTIR spectra of bioactive compounds are usually so unique
that they are called as a molecular “fingerprint.” Once the isolation of bioactive
compound is possible, then drying of extract and isolates using rotary evaporator
is done. Dried powdered plant extract spectrum can be obtained from FTIR. FTIR
software contains library of known compounds, and thus, the spectrum of an
unknown compound can be identified by its comparison. Preparation of samples
for FTIR analysis can be done in different ways. In earlier years, solid herbal plant
extract powder was milled with potassium bromide (KBr) with good trituration
techniques and then compressed into a thin pellet, which can be analyzed. Now due
to new advancements, only solid or liquid sample is available and you only have to
place one drop or one pinch of sample between two plates and the drop or sample
forms a thin film between the plates. It is the easiest way for performing FTIR, and
graphs and wave number are recorded by using computer-based software. The
region in IR spectrum above 1200 cm−1 shows spectral bands or peaks due to the
vibrations of individual bonds or functional groups under examination. The region
below 1200 cm−1 is known as the ‘fingerprint region.’ It indicates bands due to the
vibrations of the complete bioactive molecule. Complexity of compounds is seen in
fingerprint region. Intensities of the various bands in FTIR are recorded specifically
on a simple scale as strong (S), medium (M), or weak (W). And as per new tech-
niques developed, the advanced instruments of company bruker, jasco has made
easier by application of one drop or pinch of sample on the instruments and this
software will give the results. Lastly, the advantage is that samples can be reused.

8.2 Mass spectrometry (MS)

Mass spectrometry plays a vital role and works as a powerful analytical tech-
nique. It is the only technique used for identification of unknown compounds for its
molecular weight. Thus, the quantification of known compounds and elucidation
of the structure and chemical properties of molecules are possible due to MS. The
most powerful MS spectrum gives an idea about the molecular weight of sample,
which can be determined. The value of the technique is that it requires only micro-
gram amounts of material and that it can provide an accurate molecular weight and
that it may yield a complex fragmentation pattern, which is often characteristic
of that particular compound. This technique works successfully for the structural
elucidation of herbal extracts and organic compounds, for peptide or oligonucle-
otide sequencing. MS helps in monitoring the characterization of compounds in

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complex mixtures with a high specificity by defining both the molecular weight and
a diagnostic fragment of the molecule simultaneously. Gas chromatography equip-
ment can be directly coupled with rapid scan mass spectrometer (GCMS) of various
types. High resolution analysis can be performed due to coupling of equipment.
Liquid chromatography–mass spectroscopy (LC–MS) offers accurate determi-
nation of molecular weight of proteins and peptides. Isotopes pattern can also be
detected by this technique. Recent advances include electrospray, thermospray, and
ionspray ionization techniques, which offer unique advantages of high detection
sensitivity and specificity.

8.3 Nuclear magnetic resonance spectroscopy (NMR)

Nuclear magnetic resonance spectroscopy gives physical, chemical, and biologi-

cal properties of matter. C13 NMR is used to identify the types of carbon present
in the compound. H1-NMR is used to find out the types of hydrogen present in the
compound and to find out how the hydrogen atoms are connected. Proton NMR
spectroscopy basically works on principle by measuring the magnetic moments of
its hydrogen atoms and it provides a method for determining the structure of an
organic compound. In almost all compounds, hydrogen atoms are present, which
are attached to different groups such as -CH2-, -CH-, -CHO, -NH2, -CHOH-, etc.
The graphical representation of proton NMR spectrum provides a record of the
number of hydrogen atoms in these different situations. It gives information only
about the number of hydrogen atoms in the compound but not the number of
carbon atoms. However, direct information on the nature of the carbon skeleton
of the molecule can only be obtained by carbon 13 NMR spectroscopy. 13C-NMR
spectroscopy works hand in hand with proton NMR and thus the combination
of the results of two methods provides very useful information for identification
of unknown compound. It is a powerful means of structural elucidation for new
terpenoids, alkaloids, or flavonoids. It is also useful in the identification or analysis
of glycosides, in indicating the linkage between sugar moieties and their configura-
tions. Many proteins or other macromolecules can be identified by both proton and
13C-NMR. For NMR analysis, very small amount of sample is needed for analysis
and that sample can be reused for further analysis. For examples, NMR instrument
cost much so there are many sophisticated analytical instrumentation technical
analysis are available to perform your research work. Scientist handling NMR has
good hands on working of NMR. In order to get high resolution, the new technique
is liquid chromatography–nuclear magnetic resonance (LC-NMR). It is a combina-
tion of chromatographic separation technique for isolation of active fraction and
its number of hydrogen or carbon atom identification with NMR spectroscopy. It is
one of the most powerful and time-saving method for the separation and structural
elucidation of unknown compounds and mixtures, especially for the structure elu-
cidation of herbal plant extracts and their isolates in herbal natural drug discovery.

8.4 Development of new technologies in natural drug discovery research

Nature is a God’s gift for finding new herbal drugs for carrying out research
scientifically. A new driving force for screening of novel drugs, biologically active
metabolites from these products derived from nature which leads to the success
of drugs. The chemistry is a branch where the new technologies are emerging in
which pharmaceutical chemistry is very important because it deals with the health
of common people. Combinatorial chemistry, high-throughput screening, bioinfor-
matics, proteomics, and genomics are newer techniques that have emerged widely
in the field of pharmaceutical discovery research. All drug discovery research and

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technologies have enormous potential to make use of the chemical and natural
diversity of products. Newly developed techniques are growing rapidly with
good outputs in natural drug discovery [20]. These include molecular diversity,
­compound-library design, protein 3D structures, NMR-based screening, 3D-QSAR
in modern drug design, physicochemical concepts, and computer-aided drug design
using different software, its prediction of drug toxicity, and metabolism [21]. New
approaches to improve and accelerate the joint drug discovery and development
processes are expected to take place mainly from the innovation in drug target
elucidation and lead structure discovery. Powerful new technologies are revolution-
izing drug discovery. Some software will be useful in performing studies, which are
freely available such as Zinc.docking.org. It is used for calculating SWISSADME,
drug properties, SMILES formula, drug likeness properties, and many more [8].
Technologies for drug discovery advanced and diversified greatly [22]. NPDD
(natural product drug discovery) activities work hand in hand and make use
strongly with HTS, combinatorial chemistry, and genomics. New approaches have
proved to show improvement and accelerate the joint drug discovery and develop-
ment processes. New techniques are emerging and take place mainly from the
innovation in drug target elucidation. It finally leads to lead structure discovery.
Powerful new technologies are revolutionizing natural herbal drug discovery.

8.5 High-throughput screening

High-throughput screening (HTS) is a specially deigned technique in herbal

drug discovery that is a standard method for hit discovery based on identification
through stored libraries. HTS is relevant to the fields of biology and chemistry and
helps for scientific experimentation especially used in drug discovery.
HTS is using data processing and control software and sensitive detectors that
help researchers to carry out the research scientifically for designing and develop-
ing new structure from herbal drug discovery. It is robotics and allows a researcher
to quickly conduct various biochemical, genetic, or phytopharmacological tests.
Through HTS, one can rapidly identify bioactive compounds that can be useful
in a particular biomolecular pathway in inhibiting the diseased condition. Thus,
biomolecular pathway provides information about the mechanism of drug as well as
internal way of diseased condition person. The knowledge and the results of these
experiments provide starting points for designing a drug and for understanding the
interaction or role of a particular biochemical process in biology.
HTS is a relatively recent innovation and it requires high-speed computer
technology. It works on the principle of high-throughput screening of large amount
of natural compounds using computer-based technology, which is more easy and
more time saving. Knowledge can be further utilized for new herbal drug discovery.
Interest of research can be generated in natural drug discovery through all these
newer techniques. Many well-developed countries have highly specialized and
expensive screening labs to run an HTS operation. However, the countries having
interest of working in research that cases a small-to-moderately sized research
institution will use the existing HTS facility as per their convenience rather than
full set up.

9. Conclusion

With growing interest in herbal drug development with minimum side effects,
there are better opportunities to explore the medicinal and other biological proper-
ties of previously inaccessible natural products. To establish its usefulness, it is

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Pharmacognosy - Medicinal Plants

mandatory to focus on visualization and identification of unused herbal plants over

the world. Then, it is emphasized on extraction, its isolation, and characterization
of phytochemicals, which is a gift of nature in a rational and scientific way. There
is an unmet need for utilization of the natural products for the benefit of human
kind and development of new lead for drug discovery. Once the phytochemical is
obtained, this can be used for further exploration through QSAR studies, molecular
modeling, and animal studies followed by clinical trial. The success of natural
products in drug discovery essentially for pharmaceutical companies and research
institutes is essentially related to their ability and benefits to common person that
is socio-economic benefits for well-being of common person its health is important
for the world rather than all coming come to your hands if health is top priority.
Natural products contain complex chemical structures, which differ according to
their various species in nature, and when the existing high technology methods
that are available are applied, it can lead to new discovery of drugs, benefitting
the whole world. Thus, the world is always gifted with nature, and man is gifted
with brain, so let us make use of it to discover new entities that will be available to
common people in economical rate and we will be happy to lead a life on this earth.
Moreover, natural products have been, and will be, important sources of new phar-
maceutical compounds. Many years ago life was made possible or was prolonged
only due to natural herbs as per the references that can be obtained in literature. In
the new era of twenty-first century, no life is possible on earth without herbal drugs
or products that are obtained through natural herbal drug discovery. Hats off to it!

Author details

Akshada Amit Koparde1*, Rajendra Chandrashekar Doijad2

and Chandrakant Shripal Magdum3

1 Department of Pharmaceutical Chemistry, Dean Academics, KIMSDTU’s Krishna

Institute of Pharmacy, Malkapur, Karad, India

2 DEAN, KIMSDTU’s Krishna Institute of Pharmacy, Malkapur, Karad, India

3 Guide and principal, KES’s Rajarambapu College of Pharmacy, Kasegaon, India

*Address all correspondence to: akshadakakade@yahoo.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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DOI: http://dx.doi.org/10.5772/intechopen.82860
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